Recei ed: 15 Sep embe 2022
|
Accep ed: 5 Decembe 2022
DOI: 10.1002/mbo3.1342
ORIGINAL ARTICLE
Assessing pH‐dependen ac i i ies o i ulence ac o s
sec e ed by Candida albicans
Asie Ramos‐Pa do
1
|Rocío Cas o‐Ál a ez
1
|Guille mo Quindós
1
|
Elena E aso
1
|Elena Se illano
1
|Vladimi R. Kabe din
1,2,3
1
Depa men o Immunology, Mic obiology
and Pa asi ology, Uni e si y o he Basque
Coun y UPV/EHU, Leioa, Spain
2
IKERBASQUE, Basque Founda ion o
Science, Bilbao, Spain
3
Resea ch Cen e o Expe imen al Ma ine
Biology and Bio echnology (PIE‐UPV/EHU),
Plen zia, Spain
Co espondence
Elena Se illano and Vladimi R. Kabe din,
Depa men o Immunology, Mic obiology and
Pa asi ology, Uni e si y o he Basque
Coun y UPV/EHU, Leioa, Spain.
Email: [email p o ec ed] and
[email p o ec ed]
Funding in o ma ion
Ike basque, Basque Founda ion o Science;
Minis e io de Ciencia e Inno ación; Conseje ía
de Educación, Uni e sidades e In es igación
del Gobie no Vasco; Euskal He iko
Unibe si a ea
Abs ac
Candida albicans is an oppo unis ic pa hogen ha can h i e unde ad e se
condi ions including subop imal pH, nu ien sca ci y, and low le els o oxygen. I s
pa hogenici y is associa ed wi h he p oduc ion o i ulence ac o s such as
ex acellula hyd oly ic enzymes and oxins. This s udy was aimed a de e mining he
e ec o ex e nal pH, subs a e na u e, and s ain o igin on p o ease, lipase, and
hemolysin p oduc ion. To achie e his objec i e, aga pla e assays we e pe o med
a pH 5.0, 6.5, and 7.5 wi h subs a es sui able o he de ec ion o each amily o
enzymes. Mo eo e , he s udy was conduc ed wi h 20 clinical C. albicans isola es
om blood, o al ca i y, skin, u ine, and agina. The hyd oly ic zones o med a ound
he colonies we e u he measu ed o calcula e he Ez (enzyma ic zone) indexes. We
ound ha de ec ion o p o eases in skim milk aga pla es was possible o mos
isola es only a pH 5 (80%) and pH 6.5 (75%), whe eas BSA pla es could con e
p o ease de ec ion exclusi ely a pH 5 (80%). Simila ly, he pe cen age o isola es
possessing lipoly ic ac i i ies was highe a pH 5 (90%) han a pH 6.5 (70%) and pH
7.5 (35%). In con as , hemoly ic ac i i ies we e de ec ed in all isola es a pH 6.5 and
7.5 bu no a pH 5. Fu he analysis e ealed ha some di e ences in he de ec ed
ac i i ies could po en ially be a ibu ed o he ana omical o igin o hese isola es.
Collec i ely, hese indings sugges ha he pH o he si e o in ec ion migh be
c i ical o mimicking he mic oen i onmen employed o expe imen ally disco e
he key i ulence ac o s.
KEYWORDS
Candida pa hogenici y, clinical isola es, hyd oly ic zones, sec e ed enzymes
1|INTRODUCTION
The Candida genus is widely p esen in mammals' mycobiome.
Al hough many Candida species belong o he common skin and
mucosa mycobiomes (Ha p e al., 2020), hei in e ac ion wi h he
hos can occasionally igge he in ec ious disease known as
candidiasis. This disease o en occu s in immunocomp omised
pe sons o hose su e ing om dysbiosis (O ega‐Ri e os e al.,
2017; Raesi Vanani e al., 2019), especially, when he na u al
esis ance o in ec ions and/o physiological condi ions o he hos
Mic obiologyOpen. 2022;e1342. www.Mic obiologyOpen.com
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This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium,
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© 2022 The Au ho s. Mic obiologyOpen published by John Wiley & Sons L d.
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canno neu alize he ac ion o ungal i ulence ac o s, hus making
i possible o he commensal Candida o ac as an oppo unis ic
pa hogen (Ma cos‐A ias e al., 2011).
Al hough he e a e mo e han 150 Candida species, candidiasis is
mos equen ly caused by Candida albicans,Candida glab a a,
Candida opicalis,Candida pa apsilosis,o Candida k usei (Hen iques
& Williams, 2020; Quindós e al., 2018; Tu ne & Bu le , 2014). These
pa hogenic Candida species o en elici nonin asi e candidiasis
ypically localized in he o opha yngeal ca i y, skin, aginal, and
gas oin es inal ac s. In addi ion, hese ungi can also en e he
bloods eam h ough in a enous ca he e s o by in il a ion/ ans-
loca ion h ough he in es inal mucosal ba ie , hus leading o
candidemia and o he o ms o in asi e candidiasis caused by he
dissemina ion o Candida in di e en issues and o gans. In asi e
candidiasis is pa icula ly se e e, as i is usually associa ed wi h an
inc ease in mo bidi y and mo ali y.
Candida species employ a la ge a senal o i ulence ac o s o
elici in ec ions (Whi ing on e al., 2014). Thei speci ic ole in
disease can a y and depends on he si e o in ec ion and he
mo phological s a e o he ungus. Di e en C. albicans mo phologies,
such as blas ospo e, hyphae, o pseudohyphae, can g ea ly in luence
in ec ion and disease de elopmen by (i) acili a ing cell adhesion,
pene a ion/ ansloca ion h ough he in es inal mucosal ba ie , and
dissemina ion in o he bloods eam du ing ea ly s ages o in ec ion o
(ii) by supp essing hos immune esponses (Chow e al., 2021).
Se e al molecula i ulence ac o s including adhesins, in asins,
and hyd oly ic enzymes play essen ial oles in candidiasis. The majo
hyd oly ic enzymes include p o eases, lipases, and hemolysins (Maye
e al., 2013). Among di e en p o eoly ic enzymes, aspa ic p o eases
(E.C. 3.4.23) (Kashpa o e al., 1998) belong o he bes ‐s udied ones
in Candida. The key ea u e o hese bilobed globula enzymes is he
p esence o wo aspa ic esidues and a wa e molecule in hei
ca aly ic si e (Mandujano‐González e al., 2016). Aspa ic p o eases in
Candida o m wo amilies: (i) sec e ed enzymes ha a e also known
as candidapepsines (SAP), and (ii) su ace‐exposed ones (i.e., yapsins),
which emain a ached o he cell wall. In C. albicans,10SAP genes
(SAP1 o SAP10) ha e been desc ibed so a , and he co esponding
enzymes ha e been u he g ouped acco ding o he pH ange, a
which hey mani es hei op imal ac i i ies (Aoki e al., 2011). The
op imal pH o he i s g oup o enzymes (i.e., Sap2, Sap3, and Sap8)
is 2.5‐4, whe eas he second one (i.e., Sap1, Sap4, Sap5, Sap6, Sap7,
Sap9, and Sap10) is mos ac i e a pH 5–6.5. Sec e ed Candida
p o eases exe a numbe o biological unc ions including hei
pa icipa ion in he ini ial s eps o coloniza ion by damaging hos
issues and diges ion o he hos p o eins o gene a e nu ien s o
Candida. In addi ion, p o eases can help he pa hogen o escape om
he hos immune sys em by deg ading immunoglobulin A, an imic o-
bial pep ides, o inducing oxida i e bu s in he hos immune sys em
cells, he eby inducing apop osis (Rapala‐Kozik e al., 2018). Al hough
some SAPs ha e also been epo ed (Fo sbe g e al., 2019; Rapala‐
Kozik e al., 2018) in o he Candida species (i.e., C. opicalis,C.
pa apsilosis,C. dubliniensis, and Candida au is), li le is known abou
hei biochemical cha ac e is ics and biological unc ions. The amily
o yapsins, which is less cha ac e ized han candidapepsines, is widely
p esen in C. glab a a, a species ha lacks sec e ed aspa ic p o eases.
The cell‐wall‐a ached yapsins con ibu e o cell wall o ma ion and
play a signi ican ole in Candida adhesion and bio ilm o ma ion
(Rapala‐Kozik e al., 2018).
Ano he amily o i ulence ac o s includes ex acellula lipases
(EC. 3.1.1.3). Lipase ac i i y in C. albicans was i s epo ed by
We ne in 1965, and since hen, di e en s udies ha e e ealed a
leas en di e en lipase iso o ms o 20–60 kDa encoded by LIP
genes (LIP1–LIP10, espec i ely) (Hube e al., 2000). Candida albicans
lipases sha e common s uc u al elemen s ha include an α/β‐
hyd olase old, a ca aly ic iad composed o Se ‐His‐Asp/Glu, and a
cap s uc u e. Candida lipases a e s able o e a wide pH ange (4–11)
(Meh a e al., 2017) wi h op imal ac i i y a pH 7–9 (González‐
Bace io e al., 2010). Ne e heless, some lipases a e p ima ily ac i e
a mo e acidic o alkaline pH. The op imal empe a u e, a which hey
show g ea e ac i i y, is be ween 35°C and 50°C, e en hough
ce ain he mos able lipases can e icien ly p ocess hei subs a es a
empe a u es highe han 70°C (González‐Bace io e al., 2010).
Simila o p o eases, lipases play a key ole in Candida i ulence
con ibu ing o adhesion and issue damage, and nega i ely a ec ing
immune esponse, delaying in lamma o y cascade, and hus acili a -
ing Candida in asion and su i al in hos issues (Ciu ea e al., 2020).
In addi ion o he p oduc ion o p o eases and lipases, Candida
also sec e s a ious hemolysins, ini ially iden i ied in C. albicans by
Manns e al. (1994) and subsequen ly ound in o he species such as
C. opicalis (Fa e o e al., 2011) and C. pa apsilosis (Fa e o e al.,
2014). Hemolysins a e po e‐ o ming oxins ha ecognize speci ic
ligands on he su ace o he a ge cells (Gonzalez e al., 2008).
These i ulence ac o s help Candida o damage he hos cells ich in
i on, in pa icula e y h ocy es, hus p o iding access o he hos
p o eins wi h a high concen a ion o i on (e.g., hemoglobins) du ing
candidiasis (Noble, 2013). The use o aga pla es supplemen ed wi h
blood o moni o hemolysin ac i i ies made i possible o disce n wo
majo ypes o hemolysin ac i i ies. The i s , α‐hemolysis, which is
associa ed wi h incomple e lysis o blood cells due o pa ial damage
o he e y h ocy e memb ane, isually p oduces a ious sub ypes o
g eenish (o b ownish/opaque) halos a ound he ungal colony
(Sa a di e al., 2018), whe eas he second, β‐hemolysis, ep esen s
he comple e lysis o e y h ocy es e ealed by clea anslucen halos
(Hogg, 2013).
The b oad spec um and loca ion o in ec ions ha a e caused by
Candida spp. indica e ha hese pa hogens can h i e in e y di e se
en i onmen s (i.e., a di e en pH, empe a u e, access o nu ien s,
a mosphe ic oxygen, o exposu e o he immune sys em) aced by he
mic oo ganisms in he si es o in ec ion. Al hough some en i on-
men al ac o s (e.g., pH and oxygen a ailabili y) can al e he po en ial
o Candida o elici in ec ions, he ac ual impac o hese pa ame e s
is s ill poo ly cha ac e ized. The main goal o his s udy was o
in es iga e he impac o pH on he ac i i y o sec e ed i ulence
ac o s ( iz., p o eases, lipases, and hemolysins) known o hei ole
in Candida‐associa ed diseases and assess i hei ac i i y is
in luenced by he s ain o igin.
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2|MATERIALS AND METHODS
2.1 |Cul u ing and s o age o Candida isola es and
con ol s ains
2.1.1 |Candida isola es and con ol s ains
In he cou se o his s udy, 20 clinical isola es o C. albicans om i e
di e en in ec ion si es (o al ca i y, u ine, aginal ac , skin, and
blood) as well as con ol s ains we e analyzed (Table 1). The s ains
selec ed as posi i e con ols o p o ease pla e assays we e C.
albicans NCPF 3153 and C. opicalis NCPF 3111, whe eas C.
pa apsilosis ATCC 22019 and C. albicans ATCC 90028 we e used as
posi i e con ols o lipase and hemolysin assays, espec i ely. The
con ol s ains belong o he Ame ican Type Cul u e Collec ion
(ATCC) and Na ional Collec ion o Pa hogenic Fungi (NCPF) o he
Uni ed S a es and he Uni ed Kingdom, espec i ely.
2.1.2 |Candida cul u ing and s o age
All expe imen s we e pe o med wi h esh inocula p epa ed om
colonies cul u ed on Sabou aud dex ose aga (SDA; Scha lab S.L.).
The la e we e ob ained by inocula ion o pla es wi h aliquo s (ca.
30 μL) o Candida s ocks ollowed by incuba ion a 37
o
C o 24–48 h.
The cells om se e al colonies o med on SDA we e suspended in a
s e ile s o age solu ion con aining 0.6% yeas ex ac (Conda
P onadisa), 1.2% pep one (Condalab), 1.2% dex ose (LabKem), and
40% / glyce ol and we e ei he s o ed a −80
o
C in c yo ials o kep
e ige a ed a −4
o
C o daily use.
2.2 |Pla e assays o de ec ion o sec e ed
hyd oly ic and hemolysin‐like ac i i ies
Pla es o he de ec ion o sec e ed enzymes we e p epa ed as desc ibed
in Sec ion 2.2.1, inocula ed wi h con ol and es s ains, and incuba ed o
obse e hyd olysis zones (halos) a ound he colonies as de ailed in
Sec ion 2.2.2. The ac i i ies o he sec e ed enzymes we e semiquan i ied
(see Sec ion 2.2.3)bycalcula inganenzyma iczonecoe icien (Ez)based
on he a io o he diame e o he halo o he diame e o i s cogna e
colony. The expe imen s we e done in iplica e o ensu e ep oducibili y
and minimize in e ‐pla e a iabili y.
2.2.1 |P epa a ion o pla es
De ec ion o hyd oly ic and hemolysin‐like ac i i ies included he
p epa a ion o pla es wi h di e en media speci ically o mula ed o
de ec hese enzymes a di e en pH. Be o e au ocla ing, he pH o
each medium was adjus ed o pH 5, 6.5, and 7.5 using 1 M HCl o 1 M
NaOH. Then, he pH alues we e e i ied a e au ocla ing and
co ec ed (i.e., e‐adjus ed), i necessa y.
To de ec p o ease ac i i y, wo media we e p epa ed based on
he p o ocol de eloped by Cassone e al. (1987,1995). Bo h media
con ained 2% Eu opean Bac e iological aga (Condalab), 1.17% yeas
ca bon base (Sigma‐Ald ich), 0.01% yeas ex ac and we e u he
supplemen ed ei he wi h 0.2% skim milk (VWR) o 0.2% BSA (Sigma‐
Ald ich), espec i ely. In con as o he addi ion o skim milk be o e
au ocla ing, BSA was added as a 20% s e ile BSA s ock solu ion o
he second medium only a e i s au ocla ing and cooling o 50
o
C.
Lipase pla e assays we e pe o med employing a sligh ly
modi ied medium de eloped by Sli kin (2000). Following his
p o ocol, 4.5% mal aga (Sigma‐Ald ich), 5.84% NaCl, 0.056% CaCl
2
,
and 2% Tween
®
80 (Sigma‐Ald ich) we e dissol ed in dis illed wa e
and au ocla ed be o e cas ing he pla es.
Candida hemolysin p oduc ion was assessed using he pla e assay
desc ibed by Manns e al. (1994) and Luo e al. (2001) wi h some
modi ica ions. Namely, his medium con ained 6.5% SDA, 3%
dex ose, and 7% de ib ina ed sheep blood (The mo Scien i ic). The
i s wo componen s (SDA and dex ose) we e dissol ed in dis illed
wa e and pH was adjus ed be o e au ocla ing. The au ocla ed
medium was cooled down o 50
o
C and blood was added.
2.2.2 |Inocula ion and incuba ion o pla es
Be o e inocula ion, cell suspensions wi h u bidi y 0.8 McFa land uni s
(i.e., app oxima ely 10
7
cells/mL) we e p epa ed by suspending Candida
cells in a 0.85% NaCl saline solu ion (bioMé ieux S.A.). Pla es we e
TABLE 1 Candida isola es and con ol s ains used in his s udy
Species
Sou ce
O al ca i y U ine Vaginal a ea Skin Blood Con ol
Isola e/S ain ID Candida albicans 08‐052 13‐008 18‐100 05‐126 19‐002 NCPF 3153
19‐078 15‐153 18‐105 93‐432 18‐012 ATCC 90028
19‐010 07‐154 18‐135 16‐136 18‐028
18‐034 13‐028 18‐137 15‐159 18‐020
Candida pa apsilosis ATCC 22019
Candida opicalis NCPF 3111
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inocula ed by placing 10 μL o each cell suspension and hen incuba ed a
37
o
C o 5days(skimmilk‐con aining pla es), 7 days (BSA‐con aining
pla es), o 6 days (Tween 80‐con aining pla es) unde ae obic condi ions,
whe eas hemolysin de ec ion was ca ied ou a 37
o
C o 2daysinan
anae obic ja con aining CO
2
Gen™2.5 L Sache (The mo Scien i ic) o
c ea e a 5% CO
2
‐ ich a mosphe e.
2.2.3 |In e p e a ion o ac i i y halos
Measu emen s and compa isons o sec e ed enzyma ic ac i i ies
we e ca ied ou acco ding o he p ocedu e desc ibed by P ice e al.
(1982). B ie ly, he diame e s o halos and colonies we e measu ed
and u he used in his equa ion o calcula e he Ez index:
Ez Diame e o hyd olysis zone Diame e o he colon
y
=
÷
2.3 |S a is ical analysis
The da a ob ained we e s a is ically analyzed as desc ibed p e iously
(Mo ulsky, 2016) and p esen ed as g aphs by using G aphPad P ism 7.0
(G aphPad So wa e). B ie ly, an analysis o he supposed no mal
dis ibu ion o he da a was ca ied ou by pe o ming a D'Agos ino‐
F iedman es . In addi ion, pos hoc Dunn's mul iple compa isons es s
we e pe o med o es ablish i s a is ically signi ican di e ences in he
es ed ac i i ies we e obse ed a di e en pH. Bo h es s we e also
employed o assess whe he he pu a i e associa ions be ween he pH‐
dependen enzyma ic ac i i ies and s ain o igin we e s a is ically
signi ican . A e se ing he h eshold (ɑ)a 0.05,da awi hap‐ alue
lowe hano equal oɑwe e conside ed o be s a is ically signi ican .
3|RESULTS
To assess he e ec o pH on he ac i i y o sec e ed i ulence ac o s, 20
C. albicans clinical isola es (see Table 1) om a ious in ec ion si es (i.e.,
blood, o al ca i y, skin, u ine, and aginal ac ) ha di e in hei
physiological pH as well as ou con ol s ains ( wo C. albicans,andone
each C. opicalis and C. glab a a) we e used in his s udy. The ac i i y o
h ee majo sec e ed i ulence ac o s (p o eases [see Sec ion 3.1], lipases
[see Sec ion 3.2], and hemolysins [see Sec ion 3.3]) was e alua ed by
using pla e assays designa ed o sepa a ely de ec each ype o ac i i y.
3.1 |De ec ion o sec e ed p o eases
3.1.1 |De ec ion o sec e ed p o eases a di e en
pH using con ol s ains
Be o e ca ying ou pla e assays wi h clinical isola es, we es ed he
e iciency o wo di e en solid media (BSA‐and skim milk‐con aining
aga ) in he de ec ion o p o eases sec e ed by he con ol s ains a
pH 5.0, 6.5, and 7.5 unde ae obic condi ions. These expe imen s we e
done wi h 4 con ol s ains (seeTable 1) using skim milk and BSA aga
pla es incuba ed a 37°C o 5 and 7 days, espec i ely. We ound ha
only C. opicalis NCPF 3111 on skim milk aga pla es was able o
sec e p o eases de ec able a all h ee pH. In con as , sec e ion o
p o eases by C. albicans NCPF 3153 was de ec ed only a pH 6.5 and
7.5, while C. albicans ATCC 90028 yielded a p o eoly ic zone (halo)
only a pH 5. As o BSA‐con aining aga pla es, C. opicalis NCPF
3111, C. albicans NCPF 3153, and C. albicans ATCC 90028 p oduced
halos only a pH 5, while he p o eoly ic ac i i y o C. pa apsilosis ATCC
22019 was no de ec ed a any es ed pH. Mo eo e , skim milk was
he only subs a e sui able o he de ec ion o sec e ed p o eases a
pH 6.5. In e es ingly, he e was a clea di e ence in he appea ance o
halos o med on hese wo di e en media. While he halos o med on
skim milk aga pla es we e anspa en , hose on BSA‐con aining pla es
we e whi e and opaque.
3.1.2 |De ec ion o p o eases sec e ed by C.
albicans clinical isola es a di e en pH
Once he p o ocol o he de ec ion o p o eoly ic ac i i ies sec e ed by
he con ol s ains was es ed, we used i o assaying a ep esen a i e
g oup o C. albicans isola es om a ious si es o in ec ion (Table 1). The
images ob ained wi h he o al isola es a e p esen ed in Figu e 1,whe eas
he emaining da a a e summa ized in Table 2.Asseenin his able, he
assay ca ied ou wi h skim milk aga pla es e ealed ha 16 (80%) and 15
(75%)isola esshowedp o easeac i i ya pH5and6.5, espec i ely.In
con as , no p o ease ac i i y was obse ed a pH 7.5. Mo eo e , analysis
o hese da a did no e eal any signi ican associa ion be ween he o igin
o he clinical isola es es ed and hei capaci y o p oduce p o eoly ic
halos. Rega ding he assays on BSA pla es, p o ease ac i i ies we e
de ec ed only a pH 5.
Compa ison o he enzyma ic indexes co esponding o p o ease
ac i i y o C. albicans isola es es ed on skim milk aga showed s a is ically
suppo ed (p< 0.0001) di e ences in he p o eoly ic ac i i y de ec ed a
pH 7.5 and hose a pH 5 and 6.5 (Figu e 2). As o p o ease ac i i y
de ec ed on BSA‐con aining pla es (Table 2), i was obse ed only a pH
5, hus making i impossible o compa e p o eoly ic ac i i ies a di e en
pH. Acco ding o he esul s o he co ec ed Dunn's mul iple
compa isons es , we did no ind any s a is ically signi ican dependence
o p o ease ac i i y on he si e o in ec ion a any o he h ee pH alues
using ei he skim milk o BSA aga pla es (Figu e 3).
3.2 |De ec ion o sec e ed lipases
3.2.1 |Tes ing he pH‐dependen ac i i y o lipases
sec e ed by con ol s ains
Be o e using mal aga wi h pH 5, 6.5, and 7.5 a 37°C o de ec ion
o lipase ac i i y in clinical isola es, he media we e ini ially es ed
using con ol s ains. The assays we e pe o med a 37
o
Cby
incuba ing pla es o 6 days unde ae obic condi ions. Excep o C.
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RAMOS‐PARDO ET AL.
20458827, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mbo3.1342 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [15/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
FIGURE 1 De ec ion o p o eases sec e ed
by o al Candida albicans isola es 08‐052 (O1), 18‐
034 (O2), 19‐078 (O3), and 19‐010 (O4). Assays
we e ca ied ou using BSA‐con aining aga pla es
a pH 5 (a) and skim milk‐con aining aga pla es a
pH 6.5 (b) and pH 5 (c).
TABLE 2 De ec ion o sec e ed p o eases o clinical Candida albicans isola es om blood, o al ca i y, skin, u ine, and agina using
mic obiological media con aining skim milk o BSA
Subs a e
Skim milk BSA
Subs a e
Skim milk BSA
pH 5.0 6.5 7.5 5.0 6.5 7.5 pH 5.0 6.5 7.5 5.0 6.5 7.5
Blood O al ca i y
Isola e ID 18‐012 + + ‐+‐‐Isola e ID 08‐052 ‐+‐+‐‐
18‐020 + + ‐+‐‐ 18‐038 + + ‐+‐‐
18‐028 + ‐‐+‐‐ 19‐010 ‐+‐+‐‐
19‐002 + + ‐+‐‐ 19‐078 + + ‐+‐‐
Skin U ine
Isola e ID 05‐126 + + ‐+‐‐Isola e ID 07‐154 + + ‐+‐‐
15‐159 + + ‐‐‐‐ 13‐008 + + ‐+‐‐
16‐136 + + ‐‐‐‐ 13‐028 ‐‐‐+‐‐
93‐432 + + ‐+‐‐ 15‐153 ‐‐‐‐‐‐
Vagina
Isola e ID 18‐137 + ‐‐+‐‐
18‐100 + + ‐+‐‐
18‐105 + ‐‐‐‐‐
18‐135 + + ‐+‐‐
No e: The p esence and absence o de ec able p o ease ac i i ies a e indica ed by “+”and “‐,” espec i ely.
opicalis NCPF 3111, which showed no lipase ac i i y a pH 5 and 7.5,
he es o he con ol s ains yielded posi i e esul s a e e y pH.
3.2.2 |De ec ion o lipases sec e ed by C. albicans
clinical isola es a di e en pH
Following he assays wi h he con ol s ains, we nex es ed a
ep esen a i e collec ion o C. albicans s ains o lipase ac i i y
sec e ed a di e en pH. The images ob ained wi h he s ains
isola ed om he o al ca i y a e shown in Figu e 4,whe eas he
emaining da a a e summa ized inTable 3. As seen in his able, 18
(90%) and 14 isola es (70%) possessed lipase ac i i y a pH 5 and
6.5, espec i ely. In con as , only se en isola es showed lipase
ac i i y a pH 7.5. Mo eo e , he e was no e idence ha he lack
o de ec able lipase ac i i y was associa ed wi h he clinical o igin
o he lipase‐nega i e isola es. The subsequen compa ison o
lipase ac i i ies de e mined o all C. albicans clinical isola es a
di e en pH e ealed s a is ically signi ican (p< 0.0001)
di e ences in he mean alue o Ez (Figu e 5). Howe e , u he
analysis o hese da a applying he co ec ed Dunn's es did no
p o ide any s ong suppo o a dependence o hese mean
alues on he o igins o isola es (Figu e 6).
3.3 |De ec ion o sec e ed hemolysins
3.3.1 |Tes ing he pH‐dependen ac i i y
o hemolysins sec e ed by con ol s ains
Be o e ca ying ou assays wi h clinical isola es, expe imen s we e
done o se up he p o ocol using he con ol s ains, which we e
incuba ed o wo days on blood aga pla es wi h pH 5, 6.5, and 7.5 a
37°C in he p esence o 5% CO
2
. We ound ha C. opicalis NCPF
3111, C. albicans NCPF 3153, and C. albicans ATCC 90028 p oduced
hemolysis zones a pH 6.5 and 7.5. Unlike he abo e con ol s ains, C.
pa apsilosis ATCC 22019 had di icul ies g owing on blood aga pla es
and did no show any de ec able hemolysin ac i i y a all.
RAMOS‐PARDO ET AL.
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3.3.2 |De ec ion o hemolysins sec e ed by C.
albicans isola es a di e en pH
The condi ions employed o de ec he hemolysin ac i i ies o he con ol
s ains we e nex used o assay clinical isola es. A ep esen a i e se o
da a ob ained wi h o al ca i y isola es is shown in Figu e 7,whe eas he
emaining da a a e u he summa ized in Table 4. As seen in his able,
he assays ca ied ou wi h blood aga pla es p o ided de ec ion o
β‐hemolysin ac i i ies. The assays pe o med a pH 6.5 and 7.5 e ealed
hep esenceo β‐hemoly ic ac i i y in all s ains, al hough i was less
p ominen a pH7.5(Figu es7b,c). The subsequen compa ison o β‐
hemoly ic ac i i iesde e mined o allC. albicans clinical isola es a all
h ee pHs e ealed s a is ically signi ican (p< 0.0001) di e ences in he
mean alue o Ez (Figu e 8). Mo eo e , u he analysis o hese da a
applying he co ec ed Dunn's es e ealed a dependence o hese mean
alues on he o igin o hese s ains (Figu e 9). Fu he mo e, analysis o β‐
hemolysin ac i i ies disclosed s a is ically signi ican di e ences be ween
hose o aginal and blood isola es (p= 0.02) a pH 6.5 as well as be ween
hose o u ine and aginal isola es (p= 0.006) a pH 7.5.
4|DISCUSSION
Al hough Candida spp. a e condi ionally p esen in he human
mic obio a as commensal mic oo ganisms, hey can also cause
human diseases anging om mild mucosal in ec ions o se e e
deep‐sea ed ones. The ecen sp ead o candidiasis is hough o
be linked o an inc ease in he pe cen age o in asi e medical
in e en ions, he eme gence o mul i‐d ug esis an s ains, and
he g owing numbe o immunocomp omised indi iduals (Ciu ea
e al., 2020). To exe hei pa hogenic p ope ies, Candida spp.
ha e o p oduce a numbe o i ulence ac o s including sec e ed
hyd oly ic enzymes ha help pa hogens o su pass he hos
de ense and de elop diseases. Al hough C. albicans can colonize
hos issues wi h e y di e en pH, he impac o his en i on-
men al ac o on he p oduc ion and ac i i y o hyd oly ic
enzymes is la gely unknown. The e o e, his s udy aimed o
de e mine he e ec o pH on he ac i i y o hyd olases sec e ed
by C. albicans clinical isola es. To achie e his objec i e, we
analyzed he ac i i y o p o eases, lipases, and hemolysins
sec e ed by C. albicans isola es om i e di e en in ec ion si es
(i.e., blood, o al ca i y, skin, u ine, and agina) possessing di e en
physiological pH.
Whileanalyzing heimpac o pHonsec e edp o eases,we
could de ec p o ease ac i i ies only a acidic pH (i.e., a pH 5 and
6.5). These indings sugges ha he sec e ed p o eases a e likely
mo e ac i e a low pH, such as hose ound in he human agina
and skin, and he e o e migh belong o he amily o sec e ed
aspa ic p o eases p e iously iden i ied in Candida (Aoki e al.,
2011). Al hough BSA is mo e equen ly used o he de ec ion o
sec e ed p o eases, we showed ha he use o bo h subs a es
(i.e.,BSAandskimmilk)madei possible ode ec p o easeac i i y
in a highe numbe o isola es. Mo eo e , ou da a sugges ha he
use o skim milk ins ead o BSA (Table 2) could help o e eal mo e
p o ease‐posi i e isola es a pH 6.5. In e es ingly, ou pla e assays
disclosed ha some skin isola es could de elop a ilamen ous
pheno ype a pH 7.5, which is close o he pH o he blood and
in e nal issues whe e mycelial and hyphal o ms o some Candida
spp.a e ound (Yue e al., 2018). Howe e , any associa ion o he
abo e mo phological s a es wi h cu aneous candidiasis is doub ul
because he skin pH is acidic (i.e., 4.7–5.7; Ogawa e al., 1992).
Rega ding he u ine C. albicans isola es, 75% and 50% o hem
showed p o ease ac i i y in BSA‐and skim milk‐con aining media,
espec i ely. These esul s a e in conco dance wi h hose p e iously
ob ained by Alenzi (2016).
Unlike isola es om o he si es o in ec ion, o al and blood
isola es we e 100% p o ease‐posi i e on BSA pla es a pH 5. These
esul s a e consis en wi h he high pe cen age (42%–70%) o
p o ease‐posi i e o al (He nández‐Solís e al., 2014) and blood (Viei a
de Melo e al., 2019) isola es analyzed in o he s udies. Mo eo e , in
he la e case (Viei a de Melo e al., 2019), 77% o s ains mani es ed
p o ease ac i i ies a pH 3, hus sugges ing ha hey a e likely
con e ed by aspa ic p o eases ha a e no mally mo e ac i e a
acid pH.
Ano he in e es ing obse a ion was he lack o p o ease
ac i i y a pH 7.5, which closely mimics he physiological pH in he
o al ca i y (pH 7.5) and blood (pH 7.4) o heal hy indi iduals
(Table 2). This inding sugges s ha issue acidi ica ion can
po en ially s imula e p o ease ac i i ies, he eby acili a ing C.
albicans in ec ions.
FIGURE 2 Dependence o he enzyma ic p o ease ac i i y
coe icien (Ez)o Candida albicans isola es on he es ed pH o milk
aga . Sca e ed ed (pH 5), g een (pH 6.5), and blue (pH 7.5) ci cles
ep esen he s udied s ains. Ho izon al ba s ep esen he
s a is ically eliable (ɑ= 0.05) mean (longe ba ) and s anda d
de ia ion (sho e ba s) alues. B acke s indica e s a is ically eliable
se s o da a cha ac e ized by wo h esholds: **p≤0.01 and
***p≤0.001.
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RAMOS‐PARDO ET AL.
20458827, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mbo3.1342 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [15/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
(a) (b)
(c) (d)
(e)
FIGURE 3 Dependence o he p o ease ac i i y coe icien (Ez)o Candida albicans isola es on pH. Assays wi h Candida albicans isola es om he o al
ca i y (a), u ine (b), agina (c), skin (d), and blood (e) we e ca ied ou on milk aga pla es a pH 5, 6.5, and 7.5. Sca e ed ed (pH 5), g een (pH 6.5), and blue
(pH 7.5) ci cles ep esen he s udied s ains. Ho izon al ba s ep esen he mean (longe ba s) and s anda d de ia ion (sho e ba s) alues.
FIGURE 4 De ec ion o lipases sec e ed by
o al Candida albicans isola es 08‐052 (O1),
18‐034 (O2), 19‐010 (O3), 19‐078 (O4), and C.
pa apsilosis (ATCC 22019) (C). The assay was
ca ied ou a pH 5 (a), pH 6.5 (b), and pH 7.5 (c).
Thus, despi e he di e ences in physiological pH in each
in ec ion si e, no signi ican co ela ion was ound be ween he
o igin o he isola e and he co esponding pa e n o p o ease
sec e ion. Howe e , he use o di e en subs a es has e ealed
some di e ences in subs a e p e e ence. Thus, al hough C. albicans
isola es om he agina and skin we e mo e e icien in deg ading
skim milk, hose isola ed om he o al ca i y and u ina y ac
showed be e esul s wi h BSA, whe eas blood isola es we e nea ly
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equally e icien in p oducing hyd olysis zones wi h bo h subs a es
a acid pH.
Besides assaying p o ease ac i i y, he same C. albicans
isola es we e es ed o he p esence o sec e ed lipases using
Sli kin's opaci y es (Sli kin, 2000). Al hough se e al au ho s ha e
used his me hod o obse e he ac i i y o lipase‐like enzymes
(Nou aei e al., 2021; Pandey e al., 2018; Te iani e al., 2020), he
p e ious assays we e p ima ily pe o med using he s anda d
media wi h a pH close o 6.8. He e we es ed a wide ange o pH
including he physiological pH epo ed o di e en in ec ion si es
(Table 3).
Analysis o all C. albicans isola es e ealed ha 90% o hem
showed lipoly ic ac i i ies a pH 5. Howe e , he pe cen age o
lipase‐posi i e isola es was lowe a pH 6.5 (70%) and 7.5 (35%).
Te iani e al. (2020) es ed es e ase p oduc ion a pH 5 and 7,
ob aining in bo h cases a highe pe cen age (85.7%) o isola es
e icien ly sec e ing lipase‐like enzymes. In addi ion, we ound
ha he pe cen age o lipase‐posi i e o al isola es was 100% a
pH 6.5, which was consis en wi h he esul s ob ained in o he
s udies a pH 6.8 (Fa ahinia e al.,2015; Nou aei e al., 2021). In
con as , he high pe cen age (i.e., 100%) o skin isola es wi h
lipoly ic ac i i y de ec ed in ou assays a pH 6.5 was no ably
highe han ha (20%) ob ained by Skó a e al. (2010)a nea ly he
same pH (i.e., pH 6.8) using an API ZYM es . These di e ences
sugges ha he di ec de ec ion o lipase ac i i ies on aga pla es
( his s udy) can p o ide highe a es o de ec ion. As o ou esul s
ob ained a pH 6.5 wi h blood (75%), aginal (50%), and u ine
(25%) isola es, hey we e consis en wi h he high occu ence o
lipase‐p oducing s ains among C. albicans s ains associa ed wi h
blood (56.2%; Pandey e al., 2018) and di e ed om hose
(86.2%) ob ained by Za a e al. (2020) o u ine isola es. Thus, ou
TABLE 3 De ec ion o sec e ed lipases o clinical Candida albicans isola es om blood, o al ca i y, skin, u ine, and agina using mal aga
supplemen ed wi h Tween 80
Subs a e
Tween 80
Subs a e
Tween 80
pH 5.0 6.5 7.5 pH 5.0 6.5 7.5
Blood O al ca i y
Isola e ID 18‐012 + + ‐Isola e ID 08‐052 + + +
18‐020 + ‐‐ 18‐038 + + ‐
18‐028 + + ‐19‐010 + + +
19‐002 + + ‐19‐078 + + ‐
Skin U ine
Isola e ID 05‐126 + + ‐Isola e ID 07‐154 ‐‐‐
15‐159 + + + 13‐008 + ‐‐
16‐136 + + + 13‐028 + + ‐
93‐432 + + + 15‐153 ‐‐‐
Vagina
Isola e ID 18‐137 + + +
18‐100 + + +
18‐105 + ‐‐
18‐135 + ‐‐
No e: The p esence and absence o de ec able lipase ac i i ies a e indica ed by “+”and “‐,” espec i ely.
FIGURE 5 Dependence o he enzyma ic ac i i y coe icien (Ez)
o Candida albicans isola es on he es ed pH. Sca e ed ed (pH 5),
g een (pH 6.5), and blue (pH 7.5) ci cles ep esen he s udied s ains.
Ho izon al ba s ep esen he s a is ically eliable (ɑ= 0.05) mean
(longe ba ) and s anda d de ia ion (sho e ba s) alues. B acke s
indica e s a is ically dis inc se s o da a wi h wo h esholds
(*p≤0.05 and ****p≤0.0001).
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RAMOS‐PARDO ET AL.
20458827, 2023, 1, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mbo3.1342 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [15/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
(a) (b)
(c) (d)
(e)
FIGURE 6 Dependence o he lipase ac i i y coe icien (Ez)o Candida albicans isola es on pH. Assays wi h Candida albicans isola es
om he o al ca i y (a), u ine (b), agina (c), skin (d), and blood (e) we e ca ied ou on mal aga pla es a pH 5, 6.5, and 7.5. Sca e ed ed
(pH 5), g een (pH 6.5), and blue (pH 7.5) ci cles co espond o he s udied isola es. Ho izon al ba s ep esen he s a is ically eliable
(ɑ= 0.05) mean (longe ba ) and s anda d de ia ion (sho e ba s) alues. B acke s indica e s a is ically dis inc se s o da a (*p≤0.05).
FIGURE 7 De ec ion o hemolysins sec e ed
by o al Candida albicans isola es 08‐052 (O1),
18‐034 (O2), 19‐078 (O3), and 19‐010 (O4).
Assays we e ca ied ou a pH 5.0 (a), pH 6.5 (b),
and pH 7.5 (c).
da a indica e ha pH is an impo an ac o in de e mining he
p oduc ion o lipase‐like enzymes by C. albicans.Mo eo e , he
s a is ically signi ican di e ences (p< 0.0001) be ween he means
ob ained o lipase‐like ac i i ies a h ee di e en pH, sugges an
indi ec co ela ion be ween pH and lipoly ic ac i i y, as highe pH
le els esul ed in lowe enzyma ic ac i i ies. Mo eo e , u he
analysis e ealed highe ac i i ies o lipase‐like enzymes in o al
andskinisola escompa ed o hose om aginal,blood,andu ine
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